Isolation and identification of antioxidative peptides from crocodile meat hydrolysates using silica gel chromatography

Crocodiles are cultured in large numbers in Asia and other places in order to protect wild resources and meet the needs of human life. In this study, crocodile (Crocodylus siamensis) meat proteins were extracted and hydrolyzed into peptides, their antioxidant peptides were isolated and purified by silica gel chromatography and identified by LC/MS. Crocodile meat proteins were optimally extracted with water and hydrolyzed by papain based on the degree of hydrolysis and antioxidant activity. The hydrolysates were fractionated by ultrafiltration into 3 kDa, 3–30 kDa, and ≥ 30 kDa fractions. The 3 kDa fraction showed most antioxidant activity of the hydrolysates. Its active peptides were separated by silica gel column chromatography and purified by silica gel TLC, based on TLC bio-autographic assays of the activity. Four highly active peptides were identified by LC/MS as SSLTIQFVEGQFVDSYDPTIENTFTK, VPPHIY, VAPEEHPVLLTEAPLNPK, and RNGLPGPIGPAG. The identified peptides were synthesized and showed 50% free radical scavenging activities at 1.0 mg/mL, equal or higher to ascorbic acid at 0.5 mg/mL, in both DPPH and ABTS assays. The results indicated that the 3 kDa hydrolyzed peptides of crocodile meat had high antioxidant activity and the active peptides can be effectively separated and purified by silica gel column chromatography and TLC.

Protein extraction. Five grams of the defatted crocodile meat powder were extracted with 20 ml distilled water and stirred for 10, 20, 40 or 80 min, at room temperature or on boiling water bath, and then centrifuged at 1600g for 20 min. The supernatant was taken as protein extracts for enzymatic digestion.
Protein hydrolyzation. The crocodile proteins were hydrolyzed according to the previously described procedures 24 with slight modifications. The protein extracts equivalent to 5 g of crocodile meat was diluted to 100 mL with distilled water and zymolized for 2 h at their corresponding optimal pH and temperature conditions by papain (pH 6.2, 37 °C), pepsin (pH 2.0, 37 °C), trypsin (pH 7.6, 37 °C) and alcalase (pH 8.0, 50 °C). The addition of protease was 2%. After reaction, the hydrolysates were adjusted to pH 7.0 and heated at 97 °C for 10 min to deactivate the enzymes. The hydrolysates were obtained by centrifuged at 1600g for 20 min. The supernatant was freeze-dried and stored at − 20 °C. Protein analysis. Protein contents. The protein contents were determined using the Bradford assay 25 .
Degree of hydrolysis (DH). The degree of hydrolysis of the hydrolysates was determined using the TNBS (trinitro benzene sulfonic acid) method outlined in Maux et al. 26 .

SDS-PAGE and Tricine-SDS-PAGE analyses of proteins and peptides.
Crocodile meat proteins were analyzed using SDS-PAGE method according to Babini et al. 27 . Stacking and separation gels were 5% and 12%, respectively. All samples with 2% (v/v) 2-mercaptoethanol were heated for 5 min in a boiling water bath. Ten microliter of sample solution was loaded into sample well and electrophoresed at constant voltage of 80 V for stacking gel and 120 V for separating gel. The gel was stained using Coomassie Brilliant Blue G-250.
The hydrolysates were analyzed using Tricine-SDS-PAGE method, according to the method of Li et al. 28 . Stacking and separation gels were 4% and 20%, respectively. All samples with 2% (v/v) 2-mercaptoethanol were heated for 5 min in a boiling water bath. Ten microliter of sample solution was loaded and electrophoresed at constant voltage of 30 V for stacking gel and 100 V for separating gel. The gel was stained using Coomassie Brilliant Blue G-250.

Determination of the antioxidant activities.
The DPPH radical-scavenging activity was determined according to the method of Santos et al. 29 with some modification. Samples were diluted to 3, 6, 9, 12 and 15 mg/ mL with 0.01 mol/L phosphate buffer (pH 7.0). To a 160 μL aliquot of each sample, 640 μL of 0.1 mmol/L DPPH in ethanol (DPPH was dissolved in 95% ethanol) was added. The solution was well mixed and left at ambient temperature for 30 min in the dark. After that, the absorbance of the solution was spectrophotometrically measured at 517 nm. The absorbance value of the sample mixed with DPPH in ethanol was presented as A sample . The absorbance value of sample mixed with 95% ethanol solution was presented as A control . The absorbance value of DPPH in ethanol alone was presented as A blank . The percentage of DPPH scavenging activity was calculated as: Radical scavenging activity (%) = [1 − (A sample − A control )/A blank ] × 100.
The ABTS radical scavenging activity was measured according to the method of Zhang et al. 30 . Briefly, 7 mmol/L ABTS solution was well mixed with 2.45 mmol/L potassium persulfate solution in equal volume, and then the solution was left overnight (12-16 h) in the dark at room temperature. The above solution was diluted with 5 mmol/L phosphate buffer (pH 7.4) to make the absorbance by spectrophotometer at 734 nm be 0.7 ± 0.02. Twenty microliters of sample solution with 3, 6, 9, 12 and 15 mg/mL, was added to 2 mL diluted ABTS reagent, and the mixture was left at ambient temperature for 6 min accurately. Absorbance was then recorded at 734 nm. The blank was prepared in the same way, except that sample was replaced with deionized water. The percentage of ABTS radical scavenging activity was calculated as follows: Radical scavenging activity (%) = (A blank − A sample )/ A blank × 100. www.nature.com/scientificreports/ TLC-bioautography. Following the chromatographic separation and evaporation of the mobile phase, the silica gel plates were sprayed equably by chromogenic reagent according to the method of Krüger et al. 31 . Spraying ninhydrin alcohol solution (0.5%, w/v), peptide bands emerged red in 30 min at 105 °C; spraying DPPH alcohol solution (0.1%, w/v), antioxidant bands emerged white over night at room temperature; spraying copper sulfate (1%, w/v), BCA aqueous solution (5%, w/v), protein bands emerged blue over night at room temperature.
Separation and purification of peptides. Molecular weight fractionation. The hydrolysates were fractionated by using ultrafiltration membranes according to Abdelhedi et al. 32 with molecular weight cut-off (MWCO) membranes of 3 kDa and 30 kDa. The obtained fractions were < 3 kDa, 3-30 kDa, > 30 kDa. The collected fractions were freeze dried, stored at − 20 °C.
Analysis and purification of antioxidant peptide by TLC. The samples or active fractions from silica column chromatography were analyzed or purified by TLC. Silica gel TLC plates were developed/washed with methanol and then activated at 105 °C for 30 min, before used for sample separation. After loaded the samples, the TLC plates were developed with solvents of 1,2-dichloroethane: methanol:ammonia 1:9:0.2 (V/V).
Identification and synthesis of the purified peptides. Purified peptides were identified by HPLC-MS in the Wininnovate Company (Shenzhen, China). Identified active peptides were synthesized by NJ peptide Company (Nanjing, China) for activity assay.
Statistic analysis of the data. All quantitative analyses were repeated at least three times. The averages plus standard errors were presented.

Results and discussion
Preparation crocodile meat protein and its hydrolyzed peptides. Crocodile meat was homogenized and its soluble protein was extracted with water either at room temperature or on boiling water bath. As shown in Fig. 1A, the extracted protein amount was much higher at room temperature than on boiling water bath (Fig. 1A). SDS-PAGE analyses show that much more bands of protein were extracted at room temperature (Fig. 1B). There were no significant differences for extraction times from 10 to 80 min (Fig. 1A,B). Therefore, the optimal protein extraction conditions were determined to be extract with water at room temperature for 10 min. Four proteases, papain, pepsin, trypsin and alcalase, were tested for their efficiency to hydrolyze the crocodile meat proteins. Degree of hydrolysis (DH) is an important indicator in protein hydrolysis which relates to the size, the amino acid composition and the biological activity of the hydrolyzed peptides 33,34 . DH, together with SDS-PAGE analysis was used to evaluate the hydrolyzed proteins by the four proteases. As shown in Fig. 1C, DHs were lower in the hydrolysates by trypsin (DH 10.2%) and pepsin (DH 8.2%) than those by papain (DH 19.2%) and catalase (DH 17.1%). The hydrolysates by papain had the highest DH. It was reported that antioxidant activity of porcine blood plasma protein hydrolysates increased with increasing DH 33 .
The peptide size distribution in the hydrolysates by four proteases were analyzed by SDS-PAGE and tricine-SDS-PAGE. The results shown that the proteins were not well digested by pepsin and trypsin. The proteins were better but not completely digested by alcalase, and most of its peptides are between 25-35 kDa. Papain digested most of the proteins into small molecular weight peptides, and most of its peptides were less than 10 kDa (Fig. 1D). The results indicated that papain is the best enzyme to digest crocodile meat proteins and was used to make small molecular peptides.

Separation of the hydrolysates by ultrafiltration.
The protein hydrolysates of crocodile meat digested by papain were separated into three fractions by ultrafiltration based on their molecular weights (MW). Three fractions with MW less than 3 kDa (3 kDa fraction), between 3 and 30 kDa, and more than 30 kDa were obtained. As shown in Fig. 2A, the 3 kDa fraction accounted for 80% peptide amount of the hydrolysates, which is coincide with its high DH. The three peptide fractions were also analyzed by TLC, stained with ninhydrin for amino acid/peptide (Fig. 2B), or stained by BCA for protein/peptide (Fig. 2C). TLC analysis (Fig. 2B,C) showed similar results as the quantitative analysis of peptides ( Fig. 2A).
The antioxidant activities of three peptide fractions were detected quantitatively by the DPPH and ABTS methods (Fig. 2D,E). The results showed that most the antioxidant activities were found in the 3 kDa fraction and the radical scavenging activity was 52% in DPPH assays and 62% in ABTS assays. The other two fractions showed very low antioxidant with the radical scavenging activity less than 10% in both DPPH or ABTS assays (Fig. 2D,E). Mejria et al. 35 and Yang et al. 36 determined that fractions with smaller molecular weights (< 3 kDa) presented higher hydroxyl radical scavenging activity than those with higher molecular weights. Therefore, only the highly active 3 kDa fraction was selected for further experiments. www.nature.com/scientificreports/ small molecular compounds. Hoverer, it was considered not good for separation of high polar and water-soluble compounds. The SGCC was tested to isolate active peptides in the 3 kDa fraction of crocodile meat peptides. The 3 kDa fraction of peptides were dissolved in a small volume of water and loaded on the silica gel column and eluted sequentially with increased polar of solvents as described in the materials and method. The eluents were collected in fractions per BV. All eluent fractions were analyzed on TLC and their antioxidant activities were determined (Fig. 3). As shown in Fig. 3A, the peptides were well separated into 10 fractions by the silica gel column chromatography. The 10 fractions were combined into six fractions based on the TLC analysis. The combined fractions were further analyzed on TLC, with ninhydrin staining (Fig. 3B-1) and BCA staining (Fig. 3B-2), and their antioxidant activities were analyzed by TLC-bioautography (Fig. 3B-3) and by quantitative DPPH and ABTS methods (Fig. 3C). Ninhydrin staining shows amino acids and peptides (Fig. 3B-1) while BCA staining shows only peptides (Fig. 3B-2). Comparing the bands in Fig. 3B-1, B-2 with the TLC-bioautographic assay in Fig. 3B-3, the main antioxidant active bands are in the fractions 2-4 BV and most of them are peptides, rather than amino acids. Similar results were obtained in the DPPA and ABTS assays that the 2-3BV and the 4 BV are highly active and other fractions are less active. The 2-4 BV were combined together for further purification of active peptides by TLC.
The 2-4 BV from the SGCC was further separated on silica gel TLC plates. The sample was loaded on the TLC plate in line. After developed with 1,2-dichloroethane/methanol/ammonia = 3/7/0.2, the plate was cut into three pieces and stained with DPPA (left), BCA (middle) or ninhydrin (right) as shown in Fig. 4A. Four clear peptide bands can be recognized. On the separating TLC, two sides of the plate were cut off and stained with ninhydrin, and the silica gel in middle part was used for scraping off the peptide samples (Fig. 4B). The peptides in the silica gels were extracted with water and analyzed on TLC. The four peptide bands were well separated (Fig. 4C), which indicated that peptides can be effectively separated by the silica gel TLC.

Identification of active peptides by LC-MS and their activity analysis. The peptides isolated from
the TLC plate were analyzed and identified by LC-MS. The detected peptides were screened, deleting the peptides with low amount and from microorganisms (microbial contaminated) or enzyme fragments. From each active band (Fig. 4B), one peptide with high amount and originated from muscle structural proteins based on Uniprot Knowledgebase (UniProtKB) was selected as potential active peptide. The four potential active peptides The four identified peptides were synthesized for the study of their antioxidant activities. The synthesized peptides were confirmed for their amino acid sequences by LC-MS. Their antioxidant activities were quantitatively detected using ascorbic acid (0.5 mg/mL) and the crude peptide hydrolysates (1.0 mg/mL) as positive controls.
All the four peptides showed a concentration dependent antioxidant activity on the scavenging both DPPH and ABTS radicals. They had scavenging activities as low as 0.2 or 0.4 mg/mL, the activities increased with the increase of peptide concentrations. When the peptides concentration at 1.0 mg/mL, all the four peptides showed the same or higher activities as that of 0.5 mg/mL ascorbic acid. The results also showed that the radical scavenging activities of the four peptides were higher to DPPH than to ABTS radical (Fig. 5).
There are many reports on the isolation and identification peptides sequences from animal tissue. Bashir et al.

Conclusion
In this work, antioxidant peptides were prepared and analyzed, and four antioxidative peptides were purified and identified from crocodile meat hydrolysates. The results indicated that the proteins of crocodile meat were extracted with water and hydrolyzed by papain, which can make the most active peptides. The hydrolysates were ultrafiltrated into high (≥ 30 kDa), medium (3-30 kDa) and small molecular weight (≤ 3 kDa) fractions, and the 3 kDa fraction had most antioxidant activity of the hydrolysates. After separation by silica gel column chromatography and purification by silica gel TLC from the 3 kDa fraction, four highly active peptides were identified by HPLC-MS as SSLTIQFVEGQFVDSYDPTIENTFTK, VPPHIY, VAPEEHPVLLTEAPLNPK and RNGLPGPIGPAG. All the four peptides showed content dependent antioxidant activity, and can scavenge 50% or more DPPH and ABTS radicals at 1.0 mg/mL, which is equal or higher than that of 0.5 mg/mL ascorbic acid. The results indicated that the hydrolyzed peptides of crocodile meat by papain had high antioxidant activity. The peptides can be effectively separated by common silica gel column chromatography and purified by silica gel TLC, following the TLC-bioautographic assay of antioxidant activity.  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.